SB 608 
G6 D6 
1922 
Copy 1 



FACTORS INFLUENCING THE PATHOGENICITY 
OF HELMINTHOSPORIUM SATIVUM 



A THESIS 

SUBMITTED TO THE FACULTY OF THE GRADUATE 

SCHOOL OF THE UNIVERSITY OF MINNESOTA 



BY 
LOUISE DOSDALL, M.A. 



IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



JUNE, 1922 



FACTORS INFLUENCING THE PATHOGENICITY 
OF HELMINTHOSPORIUM SATIVUM 



A THESIS 

SUBMITTED TO THE FACULTY OF THE GRADUATE 

SCHOOL OF THE UNIVERSITY OF MINNESOTA 



BY 
LOUISE DOSDALL, M.A. 



IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE 
DEGREE OF DOCTOR OF PHILOSOPHY 



JUNE, 1922 



CONTENTS 









Page 

Introduction and historical review 3 

Problem 4 

Methods 5 

Source of pr.thogene 5 

Selection of host varieties 6 

Check plants 6 

Specific identity of the pathogene 7 

Temperature relations 16 

Growth of fungus on potato dextrose agar 16 

Spore germination 18 

Effect of hydrogen-ion concentration and temperature on spore germination 21 

Infection 25 

Influence of type of soil 27 

Influence of soil moisture 31 

Influence of soil fertility 39 

Comparison of several root-rot causing organisms 40 

Summrry and conclusions 44 

Literature cited 47 

ILLUSTRATIONS 

Fig. 1. Types of curves obtained from measuring length of snores of 

Helminthosporium sativum produced on potato dextrose 11 

Fig. 2. Length of spores produced on potato dextrose agar at various 

temperatures 15 

Fig. 3. Length of spores produced on different substrata at 24 C 16 

Fig. 4. Growth of H. sativum on potato dextrose agar in Petri dishes 17 

Fig. 5. Daily rate of growth of H. sativum on potato dextrose agar in Petri 

dishes 18 

Fig. 6. Percentage germination of spores in phosphoric acid-potassium 

hydroxide solutions of various hydrogen-ion concentrations 23 

Fig. 7. Percentage germination of spores in Czapek's solution minus the 

sugar at various hydrogen-ion concentrations 24 

Plate I. Helminthosporium sativum P.K.B. grown on potato dextrose agar 

at different temperatures 49 

Plate II. Helminthosporium sativum P.K.B. grown on potato dextrose agar 

at different temperatures 50 

Plate III. Marquis wheat showing effect of Helminthosporium root-rot in 

different soils 51 

Plate IV. Lion barley plants, 3 weeks old, growing on soils inoculated with 

various organisms 52 

Plate V. Lion barley plants 3 weeks old showing effect of soil organisms 

on development of root systems 53 

Plate VI. Lion barley pbnts, 2,Vz weeks old, showing effect of root infection 

by H. sativum P.K.B 54 



f 



LIBRARY OF CONGRESS 
„, I KIOUV6D 

•"May 2 4 1924 



Idocum 



ENTS DIVISION 









FACTORS INFLUENCING THE PATHOGENICITY OF 
HELMINTHOSPORIUM SATIVUM 

By Louise Dosdall! 

INTRODUCTION AND HISTORICAL REVIEW 

In 1 910 Pammel, King and Bakke (9) described a new Helmin- 
thosporium disease of barley which they called "late blight." The 
causal organism was named Helminthosporium sativum n. sp. Pammel, 
et al., had observed the disease in Iowa in 1907 and 1908. In 1909 it 
was very serious. In the same year, they report that it was also found 
in South Dakota, Minnesota, and Saskatchewan. These authors de- 
scribe the disease as follows : "Brown spots of irregular outline occur 
upon the leaves causing them to turn brown. The leaves are easily 
broken up, and in some cases completely destroyed. The disease also 
occurs upon the glumes, spikelets and seed. The straw at harvest 
is dull brown, and instead of standing erect becomes a tangled mass. 
The date of ripening of the grain corresponds with the time of full 
development of the late blight." They observed that there was con- 
siderable difference in varietal susceptibility, the degree of infection 
ranging from o to 100 per cent. Late blight was considered the most 
serious disease of barley in Iowa. 

In 1913 A. G. Johnson (7) differentiated clearly the three Helmin- 
thosporium diseases of barley in Wisconsin, and he designated the one 
caused by H. sativum P.K.B., the "American blotch disease." 

Louise Stakman (n), in 1920, showed that a Helminthosporium 
similar to the organism described as H. sativum by Pammel, King 
and Bakke, but isolated from various parts of diseased wheat and rye 
plants, was capable of causing a serious seedling blight of these hosts, 
and could also attack the older parts of the plants, namely, the leaves, 
nodes, culms, roots, glumes, and grains. In addition to wheat and 
rye, successful infections were obtained on barley and a number of 
grasses. In the spring and early summer of 1919, serious attacks of 
seedling blight caused by Helminthosporium occurred in practically all 
the wheat-growing regions of Minnesota. 

F. L. Stevens (12), also in 1920, reported that a species of 
Helminthosporium was constantly associated with foot rot disease of 
wheat in Madison County, 111. Inoculations with the organism gave 
positive results. He concluded that Helminthosporium was the cause 
of the disease. 

1 The writer wishes to express her appreciation to Dr. E. C. Stakman, under whom the 
work was done, for advice and criticism, and to Mr. M. N. Levine for his helpful criticism in 
the presentation of the biometrical studies. 



4 TECHNICAL BULLETIN 17 

In January, 1922, Hamblin (5) reported a Helminthosporium foot- 
rot disease of wheat in New South Wales, Australia. The disease 
symptoms are very similar to those of the true take-all caused by 
Ophiobolus graminis Saccardo, but there are distinguishing characters. 
Hamblin's description of the foot-rot in Australia corresponds very 
closely with that of Mrs. Stakman and of F. L. Stevens. His descrip- 
tion of the poorly developed root system with an abnormal develop- 
ment of root hairs close to the culm, giving the dead or dying root a 
"fuzzy" appearance, and the frequent growth of secondary roots above 
the first node of the affected straws, applies equally well to conditions 
observed in Minnesota during 1921. In Hamblin's opinion, the Hel- 
minthosporium disease was responsible for far more damage in 1921 in 
Australia than was the better known take-all. 

In recent years, a foot-rot disease of cereals, particularly wheat, 
rye, and barley, has been destructive in certain localities in Minnesota. 
This was especially true on certain peat lands in Anoka and St. Louis 
counties and on some of the sandy soils in Anoka, Nicollet, and 
Mahnomen counties. A Helminthosporium of the sativum type has 
been consistently isolated from the diseased plants. This organism 
is very widely distributed throughout the cereal growing region. The 
severity of its attack apparently must be greatly influenced by eco- 
logical conditions. In order to obtain more detailed and accurate in- 
formation concerning these conditions, a study of the physiology of 
the fungus, to the extent of its possible correlation with the pathogenicity 
under given conditions, was undertaken. 

PROBLEM 

In this study attention was directed primarily to the root- and foot- 
rots caused by II sativum. Little attention was given to secondary in- 
fections on leaves and heads. The soil environment was, therefore, 
of chief concern. In analyzing the factors which might influence the 
development of a disease of this type, temperature, moisture, and 
acidity would affect the growth of both the pathogene and the host,< 
and possibly also the reaction between the two. The vigor of the host 
conceivably might greatly influence the development of a disease caused 
by a facultative parasite, such as H. sativum. The type of soil in 
which they grew and the available nutriment might, therefore, change 
the balance between host and pathogene. It is difficult to separate 
and analyze the individual factors, because certain combinations intro- 
duce various complexities which are difficult to interpret. 

The following phases of the problem were investigated especially : 

1. Relation of temperature to the growth of the fungus, to spore 

germination, to infection, and to the development of the disease. 



PATHOGENICITY OF H. SATIVUM 5 

2. Relation of hydrogen-ion concentration and temperature to spore 
germination. 

3. Development of the disease in various types of soil. 

4. Influence of soil moisture on the development of the disease. 

5. Influence of soil fertilization on the development of the disease. 

6. Comparison of several root-rotting organisms. 

7. Morphological variation in the fungus with regard to its specific 
identity. 

METHODS 

SOURCE OF PATHOGENE 

During the spring and summer of 1920, tissue cultures were made 
from lesions caused by Helminthosporium on cereals and grasses. 
Twenty-two strains (isolations from various parts of different hosts 
or from different localities) of the sativum type were obtained from 
the roots, stems, nodes, leaves, and kernels of barley ; from the roots, 
stems, leaves, and kernels of wheat ; and from leaf spots of various 
grasses. Material was obtained from Anoka, Clay, Mahnomen, Nicol- 
let, Ramsey, and St. Louis counties in Minnesota ; from Tennessee, 
and from Spruce Grove and Edmonton, Alberta. 

Seven of these strains were selected for preliminary inoculation 
experiments. As a virulent root-rotting organism was desired, only 
soil inoculations were made. Four-inch pots filled with soil were treated 
with live steam for two hours on each of three successive days. Six 
pots of such soil were inoculated with each of the various strains of 
H clminthosporum. For this purpose, spores were scraped from the 
surface of potato dextrose agar cultures and mixed with water. The 
suspension of spores was poured over the soil, and the pots were in- 
cubated for several days. Three pots which had been inoculated with 
each strain were then sowed with Marquis wheat and three with 
Manchuria barley (Minn. 105). Some infection was obtained in each 
case, on both the leaves and the roots. (The check plants were slightly 
infected, as the seed had not been treated.) The plants inoculated 
with strain 82a, however, were decidedly more heavily attacked than 
the others. This was especially true of the barley plants. A Helmin- 
thosporium of the sativum type was re-isolated from lesions on both 
the barley and the wheat. A single spore culture was then made from 
the original 82a culture, and all subsequent work was done with this 
single spore strain. 

Culture 82a was originally isolated from the darkened base of 
badly stunted barley plants sent to the laboratory from the peat plots 
on the Fens experimental field, St. Louis County, Minn., in the summer 



6 TECHNICAL BULLETIN 17 

of 1920. A similar Helminthosporium was isolated from the nodes, 
sheaths, and blades of the same plants. 

In addition to H. sativum, Alternaria was frequently obtained from 
blackened kernels and nodes; a pink Fusarium was sometimes found 
on the base of the stem and roots; and Helminthosporium teres Sace. 
was occasionally isolated from the leaves and stems. 

SELECTION OF HOST VARIETIES 

In all experiments, the effect of the fungus on wheat and barley 
only was studied. In most cases where barley was tested, both Man- 
churia (Minn. 105) and Lion (Selection) were used. Manchuria is 
the barley most commonly grown in Minnesota. It is somewhat re- 
sistant to H. sativum, as shown by the work of Pammel, King and 
Bakke (9), of Hayes and Stakman (6), and of Christensen (3). For 
this reason it was used in the breeding work of Hayes and Stakman. 
It was crossed with the smooth-awned Lion, which is very susceptible 
to Helminthosporium, in an attempt to obtain a high yielding, smooth- 
awned, resistant variety. 

Marquis (Minn. 1239) was used in most of the experiments with 
wheat. In some cases Haynes Bluestem (Minn. 169) also was used. 

CHECK PLANTS 

Since it is difficult to obtain seed entirely free from Helmintho- 
sporium, especially in susceptible varieties, it was necessary to treat 
the seed in order to reduce infection in the check plants to a minimum. 
Silver nitrate was found to be the most useful disinfectant because 
the seed coats of both barley and wheat are impermeable to it (10), 
and the seed can be soaked for a long time in the solution without 
being injured. It also is more effective, especially against Helmintho- 
sporium, than mercuric bichloride. For experimental purposes, the 
method of seed treatment followed was essentially that recommended 
by Schroeder (10). The seed was dipped in 50 per cent alcohol to 
remove the air from the surface, soaked over night in X 100 silver 
nitrate solution, dipped in a dilute sodium chloride solution to precipi- 
tate as insoluble silver chloride the silver nitrate remaining on the 
surface of the seed, washed thoroly in running tap water, and dried. 
Such treatment reduced the germination of Lion barley from 90 per 
cent to 78 per cent, and of Marquis wheat from 99 per cent to 97 
per cent. 



PATHOGENICITY OF H. SATIVUM 7 

SPECIFIC IDENTITY OF THE PATHOGENE 

Three species of Helminthosporium are known to occur on barley 
in the United States. These are readily distinguished on the host by 
the symptoms. II. gramineum Rabh. causes the systemic stripe disease 
characterized by long, narrow, yellowish to brownish spots on the 
leaves and sheaths. Many spots often coalesce to form parallel stria- 
tions which run more or less the entire length of the blade and often 
down the sheath. Eventually the leaves may be reduced to shreds. 
II. teres. Sacc. and IT. sativum P.K.B. both cause local lesions which 
are characterized by peculiar blotches on the leaves. H. teres causes 
the European blotch or net blotch disease. The spots are yellowish 
brown in color, irregular in shape, and are scattered on the leaves. 
When held to the light, a characteristic net work is apparent. H. 
sativum causes the spot blotch disease characterized by irregular red- 
dish brown spots on the leaves. The spots are usually longer than 
they are broad, and, when abundant, may tend to form stripes. 

These three species also may be distinguished readily by their 
growth on potato dextrose agar. H. gramineum grows slowly, forms 
a fluffy, aerial mycelium which does not sporulate (at least not readily), 
and usually gives the medium a reddish or purplish tinge. H . teres 
also grows rather slowly. The mycelium grows very close to the 
surface of the agar. The color of the reverse side of the colony is 
greenish black. Grayish white tufts of mycelium are formed irregu- 
larly on the surface of the colony. Cylindrical, thin-walled spores are 
formed, but usually they are not abundant. In contrast to both these 
species, H . sativum grows very readily and sporulates abundantly, 
forming a flat, black or greenish black colony on agar. The abundance 
of conidia gives the surface a powdery appearance. Organisms similar 
to the one isolated from typical barley spot blotch have been isolated 
hundreds of times by workers in this laboratory from various parts 
of barley, wheat, and rye plants, and from numerous grasses. 

Pammel, King and Bakke (9) described the spores as cvlindric 
in shape, straight or curved, slender, widest at the middle, from 105 
to 130 microns in length by 15 to 20 microns in width, pale greenish 
gray to dark brown in color, with 7 to 14 cells. Later workers have 
found much shorter spores, altho observations on shape agree fairly 
well. Johnson (j) states that the spores are narrowly spindle-shaped, 
usually more or less curved. Mrs. Stakman ( 1 1 ) describes the spores 
of the organism with which she worked as either straight or curved, 
dark blue-green to brown in color, averaging 41 by 20 microns in size, 
and containing from 3 to 8 septa. Two types were isolated from dis- 
eased wheat : one a fuscous type measuring 35 by 22 microns and 
containing from 3 to 4 septa ; the other straw-colored to fuscous, 



8 TECHNICAL BULLETIN 17 

measuring 60 by 20 microns, and containing from 4 to 7 septa. Both 
of the latter are described as elliptical in shape. 

Stevens (12) makes the following statement regarding the form 
causing the foot-rot of wheat : "The spores, observed as grown on 
autoclaved wheat leaves or stems in humid air, are from 24 to 122 
microns long, the majority of them falling within the limits 80 to 90 
microns, with septa or pseudo-septa varying from o to 13, usually 
5 to 10. The spores are usually typically thickest in the region about 
midway between the base and the middle point of the spore, approach- 
ing a narrow or broadly elliptical shape, tapering somewhat toward 
each end. They possess an outer dark wall that is thin and extremely 
fragile and an inner, colorless, thick wall that is frequently soft and 
gelatinous . . . The spores usually, perhaps always, germinate 
either from one or both ends, not laterally, and are functionally only 
one-celled." 

After making a large number of isolations from H clminthosporimn 
lesions on barley, wheat, and rye, great variations were found in the 
size of the spores of the various cultures, altho they resembled each 
other more or less in shape and color. In order to find out just what 
variations might be expected in one strain, as a guide to the interpreta- 
tion of the species, a single spore was again isolated from culture 82a 
and a biometric study was made of the spores produced under various 
conditions. 

The single spore was planted on a potato dextrose agar slant and 
incubated at 24 C. for ten days. Transfers were then made to potato 
dextrose agar and to ripe autoclaved barley heads. Agar cultures were 
incubated at the following temperatures: 5 , 14 , 18 , 24 , 28 , 32 , 
and 36 C. The cultures grown at 5 and 36 did not produce spores. 
The barley head cultures were incubated at 24 C. Fresh barley 
leaves were taken from the greenhouse, placed in moist chambers, 
inoculated with spores of the same culture, and incubated at 24 . The 
length of time required for the cultures to sporulate at the different 
temperatures varied considerably; those at 24 , 28 , and 32 were 
ready for measurement in 16 days, while those at 14 required 37 days. 



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io TECHNICAL BULLETIN 17 

Spores from these various sources were then measured for length. 
In all cases, measurements were made with a Bausch and Lomh micro- 
scope, using the 4 mm. objective and an eyepiece micrometer calibrated 
so that one space was equal to 34 microns. It was observed that at 
an extreme temperature, such as 32 C, there was a great deal of 
variation in length and a large number of measurements would be re- 
quired to obtain a normal curve. Data were therefore recorded for 
the measurements of 100, 300, 500, and 1000 spores. From these data 
the mean was calculated for each group and the differences were com- 
pared in relation to the probable errors, according to the methods given 
by Babcock and Clausen (2). These data are summarized in Tables I 
and II. For 100 spores the mean was found to be 62.30 ± 0.85 microns ; 
for 300 spores 57.44 ± 0.57 microns; for 500 spores 59.66 + 0.45 
microns; and for 1000 spores 59.07 ± 0.31 microns. Their accuracy 
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ence. This borders on the verge of a significant difference, so that 
100 or perhaps even 300 spores are scarcely enough to use as a basis 
for drawing conclusions. When the mean for 500 spores is compared 
with that for 1000, the ratio is 1:1. The results obtained by measuring 
1000 spores are only very slightly more accurate than those obtained 
by measuring 500 spores. The difference is certainly not great enough 
to necessitate the measurement of the second 500 spores. 



TABLE 11 

Summary of Comparisons Between Means and Coefficients of Variability for Length of 

Spores of Helminthosporium sativum Ortainko from Measuring Populations 

of Different Size (From Data Summarized in Table I) 





Means 


Coefficients of variability 


Conditions 
compared 


Difference 


Difference divided by 
the probable error 
of the difference 


Difference 


Difference divided by 
the probable error 
of the difference 


No. of spores 


100 and 300 


4.86+1 .02 


5 


5 ■ 4 1 ± 1 • 73 


3 


100 and 500 


2.64 + 0.96 


3 


4. 66+ 1 . 31 


4 


100 and 1000 


3.23 + 0.91 


4 


4. 1 1 + 1 .24 


3 


300 and 500 


2. 22 + 0. 72 


3 


o.75±o.93 


1 


300 and 1000 


1.63 + 0.65 


3 


1 -3o±o.85 


2- 


500 and 1000 


0.59 + 0.55 


1 


0.55 + 0.68 


I 



PATHOGENICITY OF H. SATIVUM 



ii 



These comparisons are perhaps brought out more clearly by the 
curves in Figure i, in which the data obtained from measuring the 
different lots of spores have been plotted after grouping the measure- 
ments into 10 micron classes. The lowest curve, representing ioo 
spores, very clearly does not give a true index of the lower extreme 
of the total population. This explains why the mean obtained from 
ioo spores is too high. The three succeeding curves show that, as 
the number of individuals increases, the curve gradually approaches 





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Fig. 1. Types of Curves Obtained from Measuring the Length of ioo, 300, 500, and 1000 

Spores of Helminthospormm sativum Produced on Potato Dextrose 

Agar at 32° C. 



12 TECHNICAL BULLETIN 17 

a normal one. In general contour the 300-spore curve and the 500- 
spore curve approach the 1000-spore curve, altho the first is somewhat 
more irregular. The slight rise at the lower extreme indicates that 
the short spores tend to group themselves about a mode of their own. 
It is possible that improvement in the method of sampling might in- 
crease the accuracy of the results obtained from a smaller population. 
In the present study about 100 spores were measured from one mount. 
The spores were distributed as evenly as possible in the drop of water 
and each spore was measured in passing systematically over the slide 
from the upper left to the lower right hand corner. An attempt was 
made to make the mount so that two or three spores would come into 
the field at once. For all other conditions, 500 spores were measured. 

Results obtained in the study of the morphology of spores de- 
veloped on potato dextrose agar at various temperatures are interesting. 
Table III shows very little difference in the means of spores de- 
veloped at 18 and 24 . From the comparisons in Table IV it is seen 
that these differences are insignificant. If, however, we examine the 
coefficients of variability, we find that there is a significant difference 
in the amount of relative variation in the length of spores. This fact 
is very clearly brought out in the curves in Figure 2. The degree of 
variation is not increased by a temperature 4 degrees lower (14 C), 
but the mean length of the spores is slightly increased. This may be 
due to the fact that at a lower temperature the black outer wall on 
the spores and mycelium is laid down much more slowly, so that the 
spores have a longer time in which to form. This is further sub- 
stantiated by the fact that at 32 ° the spores are very much shorter. 
The amount of relative variation is practically the same as at the 
lower extreme. These differences in length of spores produced at 
various temperatures are graphically represented by the curves in 
Figure 2. 

The most striking difference in spore morphology was obtained 
by comparing the spores produced on different media. As the fresh leaf 
and the autoclaved head cultures were incubated at 24 C, we may 
compare these results with those obtained from the agar culture at 
24 C. Comparing first the spores from the head and from the agar, 
we find that the former are slightly longer. The amount of relative 
variation in the two is practically the same. On the fresh leaves, 
however, the spores are very much longer and decidedly more uniform. 
These differences are illustrated in the curves in Figure 3. 



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" 


1 


" 1 

1 


H 





i. J rf 
Pn jg Jj 



14 



TECHNICAL BULLETIN 17 



TABLE IV 

Summary of Comparisons Between Means and Coefficients of Variability for Length of 

Spores of Helnvintfwsporium sativum Produced Under Different Conditions 

From Data Summarized in Table III. 



Conditions 
compared 

14° C. and 18 C. 
14° C. and 24 C. 
14° C. and 28 C. 
14° C. and 32° C. 
18 C. and 24 C. 
18 C. and 28 C. 
18 C. and 32° C. 
24° C. and 28° C. 
24 C. and 32° C. 



28° C. and 32 C 
Head and leaves 

Head and agar 
24 C. 

Leaves and ag- r 
24 C. 

Leaves and agar 
28 C. 



Means 



Difference 


Difference divided by 
the probable error 
of the difference 


2.30 + 0.75 


3 


1 .57 + 0.66 




- 


m.34±o.6s 


17 


7.66 + 0. 71 


1 1 


0.73 + 0.64 


1 


9.04 + 0.63 


14 


5.3<5±o.68 


8 


9- 77 ±0.5 1 


19 


6.09 + 0.58 




1 1 


6.32 + 0.36 


l 1 


15.40 + 0.47 


33 


1 -99 ±0.53 


4 


1 7 -.39 ±0.47 


37 


27.16 + 0.45 




60 



Coefficients of variability 



Difference 



Difference divided by 
the probable error 
of the difference 



0.80 + 


86 

74 

83 
73 


8.62 + 


6.81+0 


2.48 + 


7.82 + 


6.01+0 


7" 
82 
62 


1 . 68 + 


1 .81 +0 


6.14 + 


69 


7-44 ±° 


73 


7-49±o 


47 


0. 04 + n 


■ 57 


7-53±o 


.48 



9 -34 ±0.52 



These facts show that in a single spore strain of a Helndntho- 
sporium of this type, marked variations may be found in the length 
of spores developed under various conditions. Differences in spore 
measurements by various authors are therefore to be expected, and 
very fine specific or varietal differences can not be drawn on the basis 
of spore size unless a large number of carefully controlled compara- 
tive studies have been made. Seemingly, the original spore meas- 
urements given by Pammel, King and Bakke are rather large (105 
to 130 microns). Stevens has come nearest to approaching this length 
with a maximum of 122 microns. The same author states that the 
majority of his spores fall within 80 to 90 microns. By examining 
Table III it will be seen that the majority of the spores developed 
upon fresh barley leaves in a moist atmosphere fall within the 80 and 
90 classes, or within 75.0 to 94.9 microns. Out of 4000 spores meas- 
ured, only 43 were longer than 100 microns. On the fresh leaves the 
longest spore measured was only 115.5 microns. However, the opti- 
mum conditions for maximum and minimum spore length have not 



PATHOGENICITY OF H. SATIVUM 



15 



necessarily been obtained in these studies. On the basis of spore 
shape and similarity with the organism obtained from typical spot 
blotch lesions and the ability to produce spot blotch symptoms on barley, 
the organism undoubtedly should be included in the species Hclmin- 
thosporium sativum P.K.B. 



0) 




Fig. 2. 



O ZO 40 60 90 /OO /20 

Length of spores (microns) 

Length of Spores of Helminthosporium sativum Produced on Potato Dextrose Agar 
at Various Temperatures 



The shape of the spores was found to be more or less the same 
under various conditions. At 24 ° and 28 , the spores tended to be 
fat, spindle-shaped to broadly elliptical, sometimes slightly curved. 
At 32 ° the thickening in the middle was less evident, and they tended 
to be more uniform in diameter. The small spores were globose to 
ovate. At 14 the longest spores were mostly narrowly cylindrical. 
There was a marked tendency for the thickened portion to occur 
nearer the base than the apex, giving the spore the shape of a slender 
flask. 



i6 



TECHNICAL BULLETIN 17 



Throughout the culture work, bizarre forms frequently appeared, 
especially forked spores which were sometimes almost stellate. Seven 
or eight such single spores were isolated and planted on agar slants. 
In each case normal spores were produced and the bizarre type occurred 
so rarely that it was quite impossible to isolate another single spore of 
the same shape from the progeny. 



240 



K 




IOO 



/20 



O 20 40 60 eo 

Length of spores (microns) 

Fig. 3. Length of Spores of Hchninthosporium sativum Produced on Different 
Substrata at 24° C. 
The solid line represents spores produced on autoclaved ripe barley heads; the line with 
short dashes, spores produced on potato dextrose agar; the line with alternate long and short 
dashes, spores produced on fresh barley leaves in a moist chamber. 



TEMPERATURE RELATIONS 

GROWTH OF FUNGUS ON POTATO DEXTROSE AGAR 

In determining the temperature relations of H. sativum, the first 
problem studied was the growth of the fungus in pure culture. In all, 
four series were run to determine the range of growth, the same gen- 
eral method being used in each. Thirty cubic centimeters of potato 
dextrose agar were poured into petri dishes 10 centimeters in diameter. 



PATHOGENICITY OF H. SATIVUM 



17 



The plates were inoculated in the center and incubated at the various 
temperatures. Each series was run in triplicate. The diameter of the 
colony was taken as the index of growth. In some cases it was impos- 
sible to control the temperature within several degrees, so that one series 
can not be checked quantitatively against the other. Different lots of 
potato dextrose also were used in the different series. The results of 
two series are given in Table V. In each case, the size of the colony 
represents the average of three plates. 



table v 

Effect of Temperature on Growth of Hclminthosporium sativum on Potato Dextrose Agar 



First Series 
(Feb. 28-March 3, 1 921) 


Second Series 
(Dec. 15-2-", 1921) 


Temperature, 
degrees C. 


Diameter of colony 
(after 9 days) 


Temperature, 
degrees C. 


Diameter of colony 
(after 7 days) 




mm. 




mm. 


0- 2 


4 


3- 6 


9 


6- 8 


14 


12-13 


18 


13-13 


28 


15-18 


30 


17-22 


42 


20-23 


77 


21-24 


5i 


27-28 


Sy 


30-32 


36 


31-33 


35 


34-35 


13 


35-39 


4 


40-42 











Fig. 4. 



Temperature ("Centigrade) 

5 IO /S 20 25 30 35 +0 

Growth of Hclminthosporium sativum on Potato Dextrose Agar in Petri Dishes 
The curve represents the diameter of the colonies at the end of seven days. 



Data obtained in the second series are shown graphically in the 
curves of Figures 4 and 5. Figure 4 shows the relative growth of 
the fungus at the various temperatures after seven days. Figure 5 



i8 TECHNICAL BULLETIN 17 

shows the daily rate of growth at each of the temperatures tested. 
Plate I shows the final appearance of the colonies in the first series, 
Plate II in the second. 

From these results we may conclude that the minimum temperature 
for the growth of Helminthosporium sativum lies near 0-2 C, the 
maximum temperature between 35 ° and 39 C. and the optimum be- 
tween 24 and 28 C. 




Time fday<s) 
o / 2 3 -? s 6 7 

fig. 5. Daily Rate of Growth of Helminthosporium sativum on Potato Dextrose Agar in 

Petri Dishes 



PATHOGENICITY OF H. SATIVUM 



iy 



SPORE GERMINATION 

In the first series of studies to determine the effect of temperature 
on spore germination, hanging drop cultures "were made on the covers 
of petri dishes, using distilled water and Czapek's solution, minus the 
sugar, as media. The spores were taken from a six-days-old bean 
agar culture. Germination counts were made after 48 hours. The 
results are eiven in Table VI. 



TABLE VI 

Spore Germination of H. sativum in Distilled Water and in Czapek's Solution at 
Various Temperatures 



Tempera- 
ture, de- 
grees C 


I St 

drop 

% 

1 — 

10 
10 
40 
68 
45 
50 



Distilled water '. 


•ii 6.7 

4th 

drop ; 

1 — 


Av. 

% 
1 — 

13 


Czapek's solution — sugar Ph 6 





2nd 
drop 


3 rd 
drop 

% 
1 — 

20 

30 
44 
48 




ISt 

drop 

% 

20 

30 
5 5 
66 
85 
77 
36 



2nd 
drop i 

% 
10 

50 

50 

86 

70 

50 




3rd 4th 
drop drop 


Av. 


1 


% 
1 — 

8 
20 
40 
41 
65 
60 




/c 
15 


% 
10 

53 

-10 


14 


6 


40 
62 


42 


14 





[ 5 

37 
58 
So 
55 



52 


18 


50 


60 

75 


59 


24 




75 


80 


30 


41 


59 


So 
42 


72 


34 




25 


38 


42 












With the exception of the results obtained at 34 C, a higher 
percentage of germination was obtained in Czapek's solution than in 
distilled water. In both cases the optimum occurred at 24 . At a 
temperature of i° C. 14 per cent of the spores germinated in Czapek's 
solution, while less than 1 per cent germinated in distilled water. At 
'42 no germination occurred in either case, while at 34 the germina- 
tion in distilled water was practically the same as at 30 , but in Czapek's 
solution a marked inhibition occurred at the higher temperature. 

After trying various methods for germinating spores, including 
hanging drops in petri dishes, in van Tieghem cells, films on slides 
in moist chambers and in moist atmosphere, the most satisfactory 
method proved to be floating the spores on the surface of a thin layer 
of a liquid medium in Syracuse watch glasses. In such cultures the 
spores can be counted directly on the surface under the low power of 
the microscope. 

In several series, through the different temperature ranges, con- 
sistently high percentages of germination were obtained at the extreme 
temperatures when water which had been redistilled over glass was 



TECHNICAL BULLETIN 17 



used. Fluctuations occurred in the different cups at any one tempera- 
ture. The results given in Table VII are typical. The percentages 
given in the table represent the average of several counts. 

TABLE VII 

Spore Germination of H. sativum in Redistilled Water at Various Temperatures 





Average percentage of germination 




Temperature, degrees C. 


Spores floating on 
surface of water 


Spores lying on 
bottom of cup 




5-5- 6.5 


87 


/6 


1 1. 5-12.0 


93 


89 


17. 0-19.0 


73 


57 


21.0-24.0 


62 


61 


2S.0-29.5 


70 


66 


30.5-32.0 


83 


46 


34.0-35.0 


65 


63 


38.0-39.0 




65 





In a second series, using hanging drops in petri dishes, 67 per cent 
of the spores germinated at 6°, 54 per cent at 12 , 79 per cent at 18 , 
91 per cent at 22 , 72 per cent at 28 , 91 per cent at 29-30 , 80 per 
cent at 32 , 82 per cent at 35 °, and 87 per cent at 39 . 

In a third and fourth series in watch crystals, the spores were not 
counted but the germination was indicated as poor, moderate, and 
good. After 24 hours incubation, in these series, the germination 
was poor at 6°, moderate to good at 12 , 18 , and 22 , and good at 
the higher temperatures. By count, 89 per cent of the spores germinated 
at 39 . At the end of 48 hours the germination was good at 6°. 

From these results with redistilled water it is difficult to detect 
any quantitative effect of temperature on the number of spores which 
germinate. Even at i° C. a small number of spores will germinate. 
This, however, is probably very near the lower limit. At the lower 
temperatures, i°, 6°, and 12°, pieces of mycelium in the cultures 
always germinated much more readily and sent out longer tubes than 
did the spores. At 40-42 no germination occurred- in the first series 
for which the results are given. In later series, however, high germi- 
nation sometimes occurred at 38-39 . Comparing these results with 
the data presented in Table V, we find 35-39 to be the maximum 
temperature for the growth of the mycelium on potato dextrose. At 
temperatures as high as 35 ° and 39 the germ tubes appeared very 
quickly, but were always short and did not increase much in length 
after two or three days. On the other hand, at 22 , 28 , and 32 , 



PATHOGENICITY OF H. SATIVUM 21 

the tubes formed such a mat of mycelium by the end of 24 hours that 
it was often difficult to determine the percentage of germination. At 
the lower temperatures a longer time was required for the germ tubes 
to appear, and they increased in length very slowly. 

In redistilled water, therefore, spores of H. sativum germinate 
about equally well at temperatures from 6° to 39 C. No very 
definite optimum temperature for germination is apparent. The char- 
acter of the germ tubes and the length of time in which they appear, 
however, would indicate that an optimum temperature lies between 
22 C. and 32 C. From these results it would seem that for the 
above-ground parts of the host, temperature is not a limiting factor 
in infection so far as spore germination is concerned. 

From the data in Table VII it will be seen that in most cases the 
percentage of germination of the submerged spores is only slightly less 
than that of those on the surface. In all cases, however, the germ 
tubes produced under water were very short and abnormally branched 
in cqmparison with the long straight tubes produced on the surface. 

The germ tube first appears as a hyaline tip at the apex of the 
spore. It is difficult to determine whether the tube breaks through 
the wall or emerges through a pore. After the tube has increased in 
^ize, the delicate exospore is split, sometimes for a third of the length 
of the spore. A second tube soon appears at the base of the spore, 
just to one side of the scar where the spore was attached to the! sporo- 
phore. The connection between the two tubes is continuous through 
the spore, showing the false nature of the septation in the endospore. 
The endospore is frequently drawn away from the exospore and forms 
a constricted tube through the latter. Two germ tubes are not always 
formed from each spore. In one lot of spores germinated in redis- 
tilled water at 22 C, it was found that 52 per cent of the spores pos- 
sessed germ tubes at both ends and 39 per cent at only one end. Nine 
per cent of the spores did not germinate. Very rarely lateral tubes 
are found. One spore was observed with a lateral germ tube from 
each of five adjacent cells at one end of a ten-celled spore. In a few 
other cases, one or two lateral tubes were observed, usually arising 
from central cells. Fusions between germ tubes are very common. 

EFFECT OF HYDROGEN-ION CONCENTRATION AND TEMPERA- 
TURE ON SPORE GERMINATION 

As so many spores always germinated in redistilled water at the 
various temperatures which permitted germination at all, the effect of 
hydrogen-ion concentration on germination was studied. Culture solu- 
tions, based on Clark and Lub's (4) titration curve for ortho-phosphoric 
acid, were made by adding varying quantities of n/5 KOH to 50 cc. 



22 



TECHNICAL BULLETIN 17 



n/5 H 3 P0 4 to give a series of hydrogen-ion concentrations ranging 
from pH 2.4 to pH 12. The pH value of the solutions was deter- 
mined colorimetrically, except for the highest three alkaline solutions 
for which the theoretical value according to the curve is given. Spores 
from a seven-weeks-old barley head culture were dusted over the 
surface of the solutions in Syracuse watch glasses. The percentage 
of germination was determined after 18 hours and after 36 hours. 
Similar series were run in triplicate at 19 , 24 , and 32 C. The 
results are given in Table VIII. 



TABLE VIII 

Spore Germination of H, sativum in H 3 PO4 — KOH Solutions of Various Hydrogen-ion 

Concentrations at Different Temperatures 





Hours 


Germination 


pH 




19° 


C. 






24° 


C. 






32° 


C. 








1 


2 


3 


Av. 


1 


2 


3 


Av. 


1 


2 


3 


Av. 






% 


% 


% 


% 


% 


% 


% 


% 


% 


% 


% 


% 


2.4 


18 








































36 


3 


4 



1 

4 


3 

2 




5 



6 












8 


2 
10 





1 


3-4 


18 


2- 


10 


9 




36 


2 


4 


7 


4 


4 





2 


2 


10 


13 


7 


10 


4-4 


18 


15 


17 


14 


15 


40 


36 


28 


35 


44 


44 


28 


39 




36 


22 


19 


14 


18 


41 


33 


id" 


30 


31 


53 


27 


38 


5-2 


18 


27 


28 


25 


27 


55 


38 


46 


46 


40 


42 


54 


44 




36 


28 


33 


25 


29 


52 


39 


52 


48 


44 


41 


52 


44 


6.4 


18 


31 


25 


40 


33 


50 


59 


57 


55 


47 


76 


52 


44 




36 


32 


33 


34 


33 


56 


64 


62 


61 


4i 


66 


55 


54 


7.0 


18 


40 


42 


60 


47 


/i 


71 


79 


"4 


86 


76 


72 


76 




36 


36 


45 


53 


45 


68 


65 


84 


72 


65 


74 


60 


66 


7-4 


18 


30 


39 


68 


46 


81 


75 


72 


76 


78 


80 


84 


81 




36 


34 


60 


67 


54 


85 


83 


73 


80 


72 


83 


87 


81 


7.8 


18 


55 


35 


36 


42 


83 


78 


80 


80 


88 


90 


94 


91 




36 


30 


34 
24 


35 
29 


33 


83 
72 


90 


90 


88 
67 


97 


94 


92 


94 


8.0 


18 


35 


29 


62 


68 


72 


65 


74 


70 




36 


35 


22 


38 


32 


76 


68 


82 


75 


75 


79 


78 


77 


8.2 


18 


21 


18 


18 


19 


60 


43 


50 


5i 


70 


80 


90 


80 




36 


19 


24 


19 


21 


72 


61 


65 


66 


75 


85 


84 


81 


9. a 


18 


25 


40 


34 


33 


62 


58 


63 


61 


87 


82 


87 


84 




36 


36 


47 


35 


39 


85 


70 


60 


72 


89 


90 


95 


9i 


1 1.4* 


18 


22 


22 


20 


21 


33 


40 


32 


35 


84 


83 


82 


83 




36 


40 


35 


36 


37 


73 


74 


82 


76 


86 


93 


90 


89 


1 1.8* 


18 


12 


18 


12 


14 








8 


3 


40 


26 


39 


35 




36 


26 


34 


37 


32 


10 





20 


to 


30 


69 


48 


39 


12.0* 


18 








1 




















1 










36 


2 


14 


1 


6 





22 





7 


9 


35 





. 15 



* Theoretical value according to Clark and Lub's titration curve for ortho-phosphoric acid. 



PATHOGENICITY OF H. SATIVUM 



23 



After an incubation of 18 hours at 19 C. no germination was 
obtained at a hydrogen-ion concentration of pH 2.4. Very slight 
germination occurred at pH 3.4; while at pH 4.4 the germination 
showed a marked increase, rising steadily until a hydrogen-ion concen- 
tration of pH 7 was reached. From this point a gradual decrease oc- 
curred, reaching the lowest point at pH 8.2. At pH 9.2 there was a 
second rise followed by a gradual falling off, until at pH 12 no germi- 
nation occurred. After incubating for 18 hours longer there was 
scarcely any change in the amount of germination on the acid side. 
There was a slight increase on the alkaline side. 

At the higher temperatures the results were very much the same 
except that the percentage of germination was increased and the point 
of maximum germination was shifted slightly to the alkaline side. 
At both 24 ° and 32 ° the optimum germination occurred on the alkaline 
side of neutrality at a hydrogen-ion concentration of pH 7.8. A much 
greater increase in germination occurred at the higher temperatures 
on the alkaline side than on the acid. The average germination after 
18 hours incubation at the different temperatures is represented by 
the curves in Figure 6. 




Hydrogen ion concentration -pH 



Fig. 6. Peixentage Germination of Spores of Hclminthosporium sativum in Phosphoric Acid- 
Potassium Hydroxide Solutions of Various Hydrogen-Ion Concentrations 



Webb (14) germinated spores of Aspergillus niger, Penicillium 
cyclopium, Fusarium sp., Botrytis cinerea, and Lenzites saepiaria in 
n/5 mannite solutions in which the hydrogen-ion concentrations were 
adjusted by the use of H 3 P0 4 and NaOH according to Clark and 
Lub's titration curve for ortho-phosphoric acid. The results obtained 
with Fusarium sp. are the only ones comparable with those obtained 
with H. sativum in the wideness of the range of hydrogen-ion con- 



24 



TECHNICAL BULLETIN 17 



centration which permits spore germination. It may be pointed out 
that both Fusarium and Helminthosporium are chiefly soil organisms. 
Among the organisms that Webb studied, only Fusarium responded 
favorably to an alkaline medium. Maximum germination occurred at 
hydrogen-ion concentrations of pH 2.8 and pH 7.4. From pH 6.2 
a steady increase in germination occurred with the increase in hydrogen- 
ion concentration up to pH 2.8. From the same point, a steady increase 
in germination also occurred with the decrease in hydrogen-ion con- 
centration and practically the same maximum was reached at a con- 
centation of pH 7.4. Examining the data of H. sativum again, there 
is a steady decrease in germination from the neutral point with the 
increase in hydrogen ions up to a concentration of pH 2.4, where no 
germination occurred during 18 hours and only very slight germina- 
tion during 36 hours. However, the usual bimodal curve is obtained, 
but, in this case, both maxima occur on the alkaline side at hydrogen-ion 
concentrations of pH 7.8 and pH 9.2. With H. sativum, germination 
occurred chiefly in the alkaline solutions. 

A series of spore germination tests was also made in Czapek's 
solution minus the sugar, with various hydrogen-ion concentrations 
ranging from pH 2.6 to pH 9.8. The results are represented by the 
curve in Figure 7. In this, case also the bimodal curve was obtained. 
The first maximum, however, occurred on the acid side of neutrality at a 
hydrogen-ion concentration of pH 6. The second was on the alkaline 
side at a concentration of pH 8. 



100 — 1 










~^^ 




















\ 


.5 
















% / 
* / 

£ / 
















I °7 

<b / 

J 

















8 



3*567 

Hydrogen ion concentration • p ri 

Fig. 7. Percentage Germination of Spores of Helminthosporium sativum in Czapek's 
Solution Minus the Sugar at Various Hydrogen-Ion Concentrations 



10 



PATHOGENICITY OF H. SATIVUM 25 

While the germination of II. sativum spores in these solutions is 
not necessarily the same as in a soil solution, certain general relation- 
ships may be pointed out. The spores will germinate through a wide 
range of hydrogen-ion concentration. Optimum germination occurs 
near the neutral point or on the alkaline side. The spores will tolerate 
high degrees of alkalinity. Germination studies in solutions more 
nearly approximating soil solutions are still desirable from the stand- 
point of a closer analysis of the development of the disease. 

INFECTION 

Marquis wheat and Lion barley were grown under sterile conditions 
in test tubes containing white sand. When the seedlings were about 
an inch high, the coleoptile was inoculated with a suspension of spores 
and incubated at various temperatures. At 22° , 25 , and 30 C. char- 
acteristic minute brown lesions were visible after 18 hours. At the 
end of five days no infection had occurred at 6° on the barley; very 
light infection was evident on the wheat. Light infection also occurred 
on both wheat and barley at 14 and 34 , and on wheat at 30 . Mod- 
erate to heavy infection occurred on both hosts at 22° and 25 , and 
also on the barley at 30 . In these cases, the typical basal browning 
characteristic of the seedling blight occurred. This was as far as it 
was possible to follow the disease under these conditions. The results 
indicate that infection will take place to some extent through a rather 
wide range of temperature from 6° to 34 C. but that for the severe 
development of the disease the range is narrower, probably 22 to 
30 . To some extent moisture, as well as temperature, was the limit- 
ing factor at the extremes. 

F. L. Stevens (13) reports that, "In an adaptation of the rag-doll 
seed tester, which allows the use of seedlings under aseptic conditions 
and variations of moisture and temperature as desired, inoculation by 
spores of Helminthosporium upon the uninjured sheath was followed 
within 24 hours by entrance of the mycelium into the host cells, and 
within 48 hours by a browned, diseased spot visible to the naked eye. 
Subsequently, when conditions favored, the mycelium invaded the inner- 
most leaves and caused general rotting and death. When inoculated 
upon the roots, there was general invasion of the cortex with very slight 
discoloration." Stevens does not report under what conditions of tem- 
perature and moisture the disease developed best. 

An attempt was made to arrive at the temperature relations gov- 
erning leaf infection by inoculating fresh excised leaves with spores 
of H . sativum, placing them in moist chambers and incubating them at 
various temperatures. After incubating for J2 hours at 6° C, both 



26 TECHNICAL BULLETIN 17 

inoculated and uninoculated check leaves were dark green, turgid, and 
normal in appearance. No signs of infection were apparent. Micro- 
scopic examination showed that many spores had germinated but so 
far as could be detected from free hand sections, the germ tubes had 
not penetrated. After the same incubation period, at 12 C, very small 
blue-green water-soaked areas were visible at the points of inoculation. 
The remainder of the leaf tissue and the uninoculated check leaves 
were still green and normal in appearance. These water-soaked areas 
were not yet visible at the end of 48 hours' incubation. At 18 C, by 
the end of the third day, there were green water-soaked areas on 
which conidiophores were beginning to appear on the inoculated leaves. 
The tissue of the leaves was still firm and the cells were turgid. While 
the infected areas retained a dark blue-green color, the rest of the 
leaf was yellow. The uninoculated check leaves were light green to 
yellow in color. After *]2 hours' incubation at 23 , 27 , and 30 C, 
there were large dark green blotches of infected tissue covered by a 
velvety mass of conidiophores. The leaf tissue was beginning to soften 
and the check leaves and non-infected areas were yellow. In the in- 
fected areas the cells were beginning to disintegrate, but the chloroplasts 
were still green. At 34 C. small water-soaked areas, 3 or 4 mm. in 
diameter, were apparent after 36 hours' incubation. At this time the 
border was beginning to turn brown. By the third day, there were small, 
brown, definite leaf spots, similar to the normal lesions produced on 
leaves in the greenhouse and in the field. The remainder of the leaf 
tissue and the check leaves were yellow. 

Under the conditions just described, there was always an abundance 
of moisture, so that the difference in reaction must have been due to the 
influence of temperature on host and fungus. During the first 36 
hours the results were probably more or less comparable to results 
obtained in growing leaves attached to the plant ; during the second 
36 hours, at some temperatures at least, the relationship was probably 
saprophytic. The most that can be claimed for results obtained in this 
way is that they are only indicative of what may happen on growing 
plants. 

The results obtained from these experiments would indicate that 
at temperatures of from 18 to 30 C, penetration into the leaf will 
take place about equally well in the presence of sufficient moisture. 
Below 24 the spots increase in size more slowly, above 24 more 
rapidly. At 12° a much longer incubation period is necessary for the 
development of water-soaked areas than at higher temperatures. At 
6° no visible infection was obtained. At a temperature as high as 34 , 
on the other hand, the development of the spots and the browning of 



PATHOGENICITY OF H. SATIVUM 27 

the host tissue occurred so rapidly that further development of the 
fungus was checked. 

While no control experiments were made with soil or leaf infection 
on growing" plants, results obtained in the greenhouse agreed in general 
with those obtained on the temperature relations of the fungus. When 
the average temperature was between 75 and 85 F., much better infec- 
tion was obtained than when the average was lower. Better results 
were obtained on an inner bench over the steam pipes than on an outer 
bench next the outside wall on the west end of the house where it 
was always cool, and vigorous plants developed in spite of heavy soil 
inoculation. 

These results also agree with those reported by McKinney (8). 
He says, "Controlled soil temperature experiments, conducted in the 
'Wisconsin temperature tanks,' and field experiments show that seedling 
infection in both spring and winter wheat and in spring barley is great- 
est at relatively high temperatures. The optimum temperature appar- 
ently lies between 26° and 28 C. This is very near the optimum rate 
of growth of H. sativum in pure culture." 

INFLUENCE OF TYPE OF SOIL 

The statement has already been made that particularly severe infec- 
tions of Hchn'uitlwspormm foot- and root-rots were observed during 
the summer of 1920 on sandy soils and on peat soils in certain localities 
in Minnesota. Consequently one of the hrst tests undertaken was a 
study of the development of the disease in different types of inoculated 
soil in order to gain, if possible, an insight into the individual factors 
which might be influencing the situation. 

A heavy loam, a sandy loam, a sand, and a peat soil were selected 
for use. The heavy loam was a black dirt used without modification ; 
the sandy loam was obtained by mixing two parts of the heavy loam 
with one part of quartz sand ; and the sandy soil by mixing one part 
of the heavy loam with two parts of coarse sand. All this soil was 
passed through a 5-millimeter mesh screen before being packed into 
the pots. The peat was a high-lime peat obtained from Anoka County 
through the Division of Soils, and fertilized according to directions 
with acid phosphate and potassium chloride to secure maximum yield 
from this particular type of soil (1). 

Small pots of steam-sterilized soil were planted with Marquis wheat 
and Lion barley. After the seeds were planted, the soil was watered 
several times with a heavy suspension of Hchninthosporium spores. 



28 



TECHNICAL BULLETIN 17 



When the plants became crowded in the small pots, they were trans- 
planted to larger pots containing sterilized soil which had been inocu- 
lated in the same way. In these pots, the plants were grown to 
maturity. 

There was no very serious seedling blight in any of the pots. The 
coleoptiles of most of the plants were darkened, and lesions were formed 
on the first leaves. The seedlings in the inoculated soils were not 
noticably smaller than those in the uninoculated, sterilized, check 
soils. When about six weeks old the height of the plants was meas- 
ured in order to determine the effect of the disease on growth. The 
results are given in Table IX. In each case the table gives the average 
height of 30 to 40 plants. 

Any differences in height of the plants in the different soils in 
either the uninoculated or inoculated series may be considered due 
to the influence of the soil in which they grew. As will be seen from 
Table IX, the differences between the plants in the different types 
of soil in the inoculated series, altho small, agree fairly well with 
similar differences in the uninoculated series. The differences between 
check plants and inoculated plants in the same type of soil may be 
considered to be the result of the disease. A comparison of the dif- 
ferences between plants in inoculated and uninoculated soils of the 
different types will give an index of the influence of the soil type on 
the development of the disease. 

TABLE IX 

Average Height of Wheat and Barley Plants Grown in Inoculated and 

Uninoculated Soils of Various Types 





Type of soil 


Marquis wheat 


Lion barley 




Inoculated 


Check 


Inoculated 


Check 


Heavy 


loam 


cm. 
22.8 

21.0 _ 


cm. 
30.2 


cm. 
24.5 


cm. 
27.6 


Sandy 


loam 


3^-3 
33-6* 


29.0 


3i-i 


Sand 


23.O* 


25-3 


28.3 


Peat 


29.4* 


41.7* 


29.4 


33-6 



* Plants were measured two days later than those in the heavy loam and in the sandy 
loam, and so can not be compared with these. 



Judging by the height of the plants at this stage, the barley de- 
veloped about equally well in the heavy loam and in the sand, the 
difference in the average height being 8 millimeters in the inoculated 
series and 7 millimeters in the check series. The increases over this 
amount were about equal in the sandy loam and in the peat, the advan- 
tage being slightly in favor of the latter. The difference between the 
height of plants in inoculated and uninoculated soils was practically the 



PATHOGENICITY OF H. SATIJ r UM 29 

same in the heavy loam and in the sand. This would indicate that these 
two types of soil had practically an equal influence on the development 
of the disease. 

The difference was less pronounced in the sandy loam, showing that 
here the disease had least influence on the size of the plants. The great- 
est difference in height was in the peat soil, indicating that here the 
disease had most influence on the growth of the plant. From these 
results it is apparent that root-rot of barley produced the greatest effect 
on the host in the peat, a less marked effect in the sand and heavy loam, 
and the least effect in the sandy loam. On the whole, the differences 
were very small. The further development of the disease on the barley 
plants was not followed. 

The conclusions to be drawn from the height of the wheat plants at 
this stage must be derived from comparisons between the differences in 
the height of diseased and check plants in the same type of soil. It is 
obvious that the effect of the disease is much more marked on the wheat 
than on the barley. The least effect of the disease on the growth of the 
plants was obtained in the heavy loam. There was practically an equal 
increase in effect in the other three types of soil. 

The wheat was then transplanted to larger pots of inoculated soil. 
In each case, the most severely diseased plants were transferred. After 
transplanting, the check plants grew much faster than the diseased 
plants, and headed several days earlier. Plate III shows the compara- 
tive vigor and size of the plants in the different types of soil at maturity. 
Final observations were made on the Marquis wheat just before the 
heads began to turn yellow. The plants were removed from the soil, 
carefully washed, and examined for foot- and root-rot. 

In the heavy loam soil, both diseased and check plants averaged 
3.5 culms per plant. While the severity of infection, measured by the 
degree of browning at the base of the plant, was moderate, there was 
very little difference in the extent of the root systems. The check 
plants headed four days earlier than the diseased plants and were con- 
siderably more vigorous. A slight browning occurred at the base of 
most of the mature check plants which resembled slightly a light infec- 
tion by Helminthosporium. The lesions, however, were less definite 
and no organism was obtained from tissue cultures. H. sativum was 
isolated from the base of diseased plants. 

In the sandy loam, the average number of culms on each diseased 
plant was 3, on each check plant 2.5. The basal infection was moderate. 
There was -little difference in the root systems. 

In sand the average number of culms on each diseased plant was 3, 
on each check plant 2. The infection at the base of the diseased plants 



30 TECHNICAL BULLETIN 17 

ranged from moderate to heavy minus. The root systems of the dis- 
eased plants were considerably less extensive, brown lesions were 
numerous, and the roots were very easily broken. The contrast between 
diseased and check plants was greatest in this type of soil. 

In the peat soil the average number of culms on inoculated plants 
was 2.7, on uninoculated 2.6. Basal infection was light to moderate. 
There was very little difference in general appearance of the plants 
grown in inoculated soil and in uninoculated soil. The best plants in 
both series were obtained in the peat soil. 

Under the conditions studied, the root-rot inhibited the growth of 
Lion barley most, during the first six weeks, in the peat soil. The 
effect of the disease was less evident in the heavy loam and the sand, 
and least evident in the sandy loam. During the same period, the 
growth of Marquis wheat was least inhibited in the heavy loam. The 
effect of the disease on the growth of the plants was markedly in- 
creased, and to practically the same extent, in the other three types of 
soil. By the time of maturity, however, the disease had developed 
much more severely in the sand, as evidenced by the smaller size of the 
plants, their decreased vigor, the amount of basal browning, and the 
breaking down of the root system. The effect of the disease was almost 
as severe in the heavy loam. In both the sandy loam and the peat 
there was only a very slight difference between the plants grown in 
inoculated and uninoculated soil. 

In analyzing the factors involved in these various soils, it may be 
pointed out that in the loam soils, in addition to the change in physical 
texture brought about by adding increasing quantities of sand to the 
original heavy loam, there has been a dilution of the mineral nutrients 
of the host, a decrease in the water-holding capacity, a decrease in the 
amount of organic matter in the soil, and an increase in the amount of 
soil aeration. All these factors may be assumed to have an influence on 
both the host and the pathogene. On the other hand, in the peat soil, 
we have a high organic content, a high water-holding capacity, and an 
optimum of mineral nutrients for the host. The abundant moisture 
and high organic content of the peat soil should seemingly be conducive 
to extensive saprophytic growth of Hclminthosporium, thus greatly in- 
creasing the amount of inoculum and the chance for infection of the 
growing host. This tendency, however, seems to be counterbalanced by 
the optimum conditions offered for the growth of the host. On the 
other hand, the greater severity of the disease in sand and heavy loam 
suggests a possible influence of the soil water. These results led to a 
further study of the influence of the soil moisture and of soil fertility 
on the development of the disease. 



PATHOGENICITY OF H. SATIVUM 31 

INFLUENCE OF SOIL MOISTURE 

Preliminary series of experiments were carried out in the green- 
house in the following manner in order to determine the effect of soil 
moisture on the development of H. sativum on Lion barley. Light loam 
soil was sifted through a 5 millimeter screen, packed into jars, and 
sterilized. The sterilized soil was mixed with a culture of H. sativum 
grown on sterilized oats seed. Five degrees of soil moisture were main- 
tained more or less uniformly by adding definite amounts of water each 
day. In the fifth series the soil was kept saturated by standing the porous 
pots in jars of water. In the other four series the soil was in glazed jars 
and the soil moisture was regulated by adding different amounts of 
water. Each moisture series was carried out in triplicate, in both inocu- 
lated and uninoculated soil. The seed was sterilized with silver nitrate 
before planting. 

Comparative results on the infection above ground at the end of 
three weeks and below ground at the end of four weeks are summarized 
in Table N. In this table the infection is designated by fractions ; the 
denominator represents the number of plants in one pot, the numerator 
the number that were infected. On examining the data, it is seen that, 
as far as the above ground parts of the plants are concerned, the per- 
centage of infection, as well as the severity, is increased as the amount 
of soil moisture is increased. Comparatively few infections occurred 
on the check plants. 

The relation of soil moisture to root infection is a little more difficult 
to see, as here the development of the roots in inoculated and uninocu- 
lated soils with the same moisture content must be compared, and then 
these differences compared for the various series. The roots were most 
severely rotted in the saturated inoculated soil, and the difference in 
the extent of the root systems of diseased and check plants was greatest 
here. The next greatest difference was in the first series, with a soil 
moisture content averaging 9 per cent, while the least difference was 
found in the third and fourth series. In these two series the plants 
grew best of all, in both the inoculated and uninoculated soils. Injury 
to the roots is brought about by rather limited local lesions which kill 
the root tips or cut off portions of the roots when the lesions occur back 
from the tips. Very often the roots are rotted off near the seed. 

These results would indicate that plants suffer most from root infec- 
tion by H. sativum in soils containing both maximum and minimum 
extremes of moisture. When conditions are more nearly favorable for 
the optimum growth of the plants, the effect of the disease can be over- 
come, and root systems are developed in inoculated soil almost equal 
in extent to those in clean soil. 



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36 TECHNICAL BULLETIN 17 

An attempt was made to check up the moisture relations of the 
disease under field conditions. In a series of six square-rod plots, three 
were planted to barley and three to wheat. The north half of the barley 
plots was planted with Manchuria and the south half with Lion; the 
north half of the wheat plots was planted with Marquis, the south with 
Bluestem. A field drill was used for planting. Several days before 
planting, the soil was inoculated by applying //. sativum grown on 
sterilized wheat seed. One half gallon of the culture was applied to 
each square rod. After the seed was planted, one of the barley plots 
and one of the wheat plots was sprinkled for ten minutes each morning 
and evening, a second of each was sprinkled for five minutes each morn- 
ing and evening, and to the third no water was added. During the 
first part of the season, very little infection appeared on any of the 
plants except Lion barley. The weather was very cold during the first 
two weeks after seeding and scarcely any infection occurred. There 
was no seedling blight on the unwatered plot, very little on the moder- 
ately watered plot, and only a moderate amount on the heavily watered 
plot. Infection occurred mainly on the above-ground parts, resulting 
in leaf spots and lesions on the sheaths. Less than 1 per cent of the 
plants were killed. After the first two weeks, the amount of infection 
increased very rapidly on the heavily watered plot, so that at the end 
of six weeks 100 per cent of the plants were infected, and most of 
them were severely attacked. On the moderately watered plot about 
80 per cent of the plants were infected, the severity of the infection 
ranging from light to moderate plus. On the unwatered plot, about 
50 per cent of the plants were infected, the severity of the infection 
ranging from light to moderate. 

The method of applying the water tended to keep a film of moisture 
on the lower, shaded leaves, forming almost a moist chamber near the 
surface of the soil. The sprinkling also offered a good opportunity for 
the spores to be splashed from the soil onto the leaves. During June, 
the weather was very hot. As a result of this combination of circum- 
stances, the plants on the heavily watered plot were literally covered 
with 77. sativum lesions. In many cases the plants were so badly in- 
fected at the base that they rotted off. This was not true to such a 
marked extent on the moderately watered plot owing, probably, to the 
fact that the surface of the soil was not kept wet enough to maintain 
a more or less constant layer of moisture just above the surface of the 
ground. The Manchuria barley was moderately affected, but the wheats 
only slightly. 



PATHOGENICITY OF H. SATIVUM 37 

Final data were taken just before the heads ripened. An attempt 
was made to obtain a quantitative estimate of the percentage of plants. 
infected for the whole plot and an average of the degree of infection 
on the roots, foot, and node from individual plants. For this purpose, 
approximately equally large groups of plants were dug from the center, 
and also from each corner of the plots, two feet from the margins. 
Ten plants were taken from each group and for these fifty plants, the 
following data were recorded : the number of culms which headed ; the 
number of tillers which did not head ; the degree of infection — indicated 
as heavy, moderate, or light on the roots, foot, and nodes. Finally, the 
seed from each plot was weighed after threshing and the yield per acre 
was calculated from this. These data are summarized in Table XI. 
In order to obtain a simple mathematical expression for making com- 
parisons, the percentage of heavy infections was multiplied by 10, the 
moderate by 5, and the light by I, and the sum was taken as the index 
of the total infection. In order more easily to make comparisons, these 
sums were reduced to unity. Finally, these expressions for root and 
foot infections were totaled to obtain a means of comparing the com- 
bined foot- and root-rot with the relative amount of soil moisture and 
with the yield. For the sake of comparison these were also reduced to 
unity. In some cases the Helminthosporium infections were so com- 
plicated by Fusarium infections that it is quite impossible to say how 
much of the damage was due to each organism. This was especially 
true on the Manchuria barley. In general, the amount of injur}- was 
small. Altho the root-rot and the basal infection as measured by the 
degree of browning was sometimes heavy on a large number of plants, 
the plants were not noticeably stunted or immature as is often the case 
in severe cases of foot-rot. On the whole, there was more foot- and 
root-rot on the barley than on the wheat. The Lion barley alone shows 
an increase in the amount of foot-rot as the relative amount of soil 
moisture is increased. The differences are so small, however, that they 
can not have much significance. There were no indications of a correla- 
tion in yield with either the relative amount of soil moisture or the 
amount of foot- and root-rot. For both varieties of wheat, foot- and 
root-rot was slightly worse on the driest plot. In no case did the rela- 
tive amount of soil moisture or infection influence the tillering of the 
plants. 



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PATHOGENICITY OF H. SATIVUM 39 

Many factors that are difficult to control enter into field experiments 
and complicate the results in such a way as to make them indicative 
rather than conclusive. In these experiments foot- and root-rot de- 
veloped slightly more vigorously on Lion barley in the wettest soil and 
on the two varieties of wheat in the driest soil. This may be only a 
confirmation of the earlier greenhouse experience that root infection 
tends to be worse under either extremely dry or extremely wet conditions. 

INFLUENCE OF SOIL FERTILITY 

The effect of soil fertilization on the development of foot- and root- 
rot caused by H. sativum was studied in field plots on Lion and Man- 
churia barley, and on Marquis and Bluestem wheat. Potassium and 
nitrogen in the form of muriate of potash and nitrate of soda were 
added to square-rod plots at the rate of 600 and 300 pounds of fertilizer 
to the acre. Treble superphosphate was added at the rate of 200 and 
100 pounds. These fertilizers were so applied that there were plots 
with a heavy and a light application of each alone and in combination 
with a heavy and light application of each of the others, except that 
there were no combinations of nitrogen and potassium. In addition to 
these, complete fertilizer was applied at the rate of 600 and 300 pounds 
and manure at the rate of 20 tons and 10 tons per acre. Unfertilized 
plots were left as checks. All the plots were run in duplicate, one series 
planted with wheat and one with barley. The north half of the wheat 
plots was planted with Marquis, the south half with Bluestem, the 
north half of the barley plots with Lion, the south half with Manchuria. 

Several days before planting, H. sativum grown on sterilized wheat 
seed was applied on the surface of the soil at the rate of one half gallon 
of the culture to the square rod. The plots were seeded with a field 
drill, wheat at the rate of 90 pounds to the acre, and barley at the rate 
of 86 pounds. This is the normal rate of seeding for this section of 
the country. 

Practically no seedling blight developed on any of the plots. Leaf 
lesions and foot-rot first appeared on the barley during the second and 
third weeks, and soon after lesions developed also on the wheat. 

There was considerable difference in the vigor and height of plants 
on the different plots in response to the different fertilizers. During 
the latter part of the season, there were differences in the amount of 
lodging on the various plots. Final data on the amount of foot- and 
root-rot were taken just previous to the ripening of the grain. In order 
to obtain an approximately quantitative expression for the amount of 
infection in each plot. 50 plants were selected from each half square 
rod, 10 from each corner, two feet in 'from the margins, and 10 from 
the center of the plot. For each of these plants the following data 



40 TECHNICAL BULLETIN 17 

were recorded : the number of culms which developed heads ; the num- 
ber of tillers which did not mature; the degree of infection (designated 
as heavy, moderate, or light) on the roots, the foot, and the nodes. 
After harvesting, the weight of the straw and of the threshed grain 
was recorded, and from this the yield per acre was calculated. The 
infection of Lion barley was slightly more pronounced than on the other 
hosts. These data for Lion barley are summarized in Table XII. 

In order to arrive at a simple factor which would express the total 
infection for the roots, the foot, and the nodes for a single plot and 
would also take into account the severity of infection as well as the 
percentage of plants infected, the number of heavy infections was 
multiplied by 10, the number of moderate infections by 5, and the num- 
ber of light infections by 1, and the three products were summed. This 
was taken as an arbitrary index of the infection. In order to make 
comparison more simple, these summations were reduced to unity by 
dividing each by the lowest sum. This is the factor designated as total 
infection in Table XII. In order to compare the combined effect of 
root- and foot-rot, the summation for each was added and these sums 
in turn were reduced to unity. 

In order to bring out the relation of infection to fertilizers and 
yields, the arbitrary indices of infection were grouped into three 
classes and the yields per acre into three classes, and the fertilizer plots 
were arranged according to their infection and yield in the various 
classes as shown in Table XIII. From this summary table it is quite 
clear that the amount of foot- and root-rot is not correlated with any 
particular fertilizer. 

The disease did not appear in its severest form on any of the plots. 
Under the conditions of this experiment, there was no evidence of 
severe stunting of the plants or of excessive tillering. 

COMPARISON OF SEVERAL ROOT-ROT CAUSING 
ORGANISMS 

In order to obtain comparative results on the pathogenic effect of 
different soil organisms on Marquis wheat and Lion barley, a culture 
of Helminthosporium isolated from the foot-rot of wheat in Illinois by 
F. L. Stevens and a culture of Fusarium culmorwm (W. Sm.) 
Saccardo isolated from scabby wheat, were compared with the Helmin- 
thosporium sativum isolated from barley foot-rot in Minnesota. 



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TECHNICAL BULLETIN 17 



These organisms were grown on sterilized wheat seed to obtain a 
large amount of inoculum. At the end of two months, the cultures 
were practically masses of mycelium and spores. These masses were 
passed through a meat grinder and the pulp was thoroly mixed with 
sterilized soil in which the wheat and barley were then planted. The 
observations on seedling injury at the end of twenty days are sum- 
marized in Table XIV. 

TABLE XIII 

Summary of Data in Table XII 

Infection classes 



Yield 


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The results on the Lion barley were very sharp. Unfortunately, 
rats molested some of the pots. In the barley series, however, only the 
check plants were injured. Three plants were left in each of the three 
pots. The comparative size of the plants grown in soil inoculated with 
the various organisms is very well shown by Plate IV. 

The figures in Table XIV show that all three of the organisms 
caused a dwarfing of the barley, the two cultures of Helminthosporium 
to a much greater extent than the Fusarium. The Minnesota strain 
almost completely destroyed the plants. The effect of the three organ- 
isms on the root systems is shown by Plate V. The nature of the injury 
caused by severe infections of Helminthosporium is further illustrated 
by Plate VI, where eight seedlings showing various degrees of infection 
are shown beside a normal seedling of the same age grown in sterilized 
soil. 



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44 TECHNICAL BULLETIN 17 

The results obtained with Marquis wheat are not so clear, as all the 
pots were molested more or less. The amount of injury caused by foot- 
and root-rot was very much less than on the barley. The Illinois strain 
of Helminthosporium seemed to cause slightly more injury than the 
Minnesota strain. 

This series was started in the greenhouse during warm weather early 
in October when the temperature in the house was very high. The sub- 
sequent development of the disease was most interesting. After three 
weeks, the plants were thinned so that only three remained in each pot 
except for those inoculated with H. sativum, culture 82a, of which only 
three plants in each pot survived. These were badly stunted and in- 
fected at the time. The pots were kept next to the outer west wall of 
the greenhouse, where the temperature was always low during winter. 
The position of the pots was changed periodically so that all the plants 
would have more or less equal advantages as to sunlight. The plants 
grew remarkably well, and after a few weeks scarcely any differences 
could be detected between the different series. The barley stooled ex- 
cessively and did not head well. The wheat was very good. At heading 
time, late in April, there was practically no difference between either 
the wheat or the barley plants grown in the clean soil and in the soil 
inoculated with the Minnesota strain of //. sativum or the Fusarium 
culmorum. The wheat in the soil inoculated with the Illinois strain of 
Helminthosporium was very bushy and developed only tine or two heads 
per pot, while the other wheats developed from four to eight. The 
Lion barley was also slightly poorer in the soil inoculated with the 
Illinois strain than in the others. The barley did not head well, how- 
ever, in any case. 

Under the conditions of this experiment, then, the Helminthosporium 
caused more injury to Marquis wheat and Lion barley than the Fusa- 
rium, both in the seeding and the mature stages. While the Minnesota 
strain of Helminthosporium caused decidedly more seedling blight on 
the barley, the Illinois strain caused slightly more stunting of the mature 
plants. The Illinois strain caused more injury to the wheat at both 
stages. 

SUMMARY AND CONCLUSIONS 

In recent years a foot- and root-rot of wheat, rye, and barley has 
been serious in certain localities in Minnesota. A Helminthosporium 
of the sativum type has been constantly isolated from the diseased 
plants. In addition to causing a foot- and root-rot, the same type of 
organism attacks the leaves and stems and especially the nodes, glumes, 
and kernels of cereals and a large number of wild grasses. A strain 
of the organism was isolated from a foot-rot of barley. A pure culture 
was secured by isolating a single spore. The morphology of the organ- 



PATHOGENICITY OF H. SATIVUM 45 

ism was studied under various conditions with regard to its specific 
identity. The physiology and pathogenesis were studied with special 
reference to environmental conditions most favorable to the develop- 
ment of foot- and root-rot. 

The organism is capable of causing disease symptoms similar to 
those described by Pammel, King and Bakke in 1910. Discrepancies 
are found between the spore measurements of this organism and that 
described by Pammel, King and Bakke, but since wide variations oc- 
curred under different conditions in a single-spore culture of the 
organism studied, the similarity of disease symptoms is considered 
sufficient justification for considering the organism to be Helminthos- 
porium sativum P. K. B. 

Variations in the morphology of the spores were found to occur 
under different conditions of growth. For spores as variable in length 
as those of H. sativum, it was found necessary to measure 500 spores 
in order to obtain accurate results. 

On potato dextrose agar, significant differences in mean length of 
the spores occur when the organism is grown at different temperatures. 
The shortest spores with a mean length of 55.981ho.35 microns were 
produced at 28 C. The longest spores, with a mean length of 
67.32zbo.55 microns, were produced at 14 C. The difference between 
the two means is 14 times the probable error of the difference. 

The greatest differences in length were found between spores pro- 
duced on different substrata. At 24 C. the mean length of the spores 
produced on potato dextrose was 65.751+10. 37 microns, on autoclaved 
ripe barley heads 67.74zto.38 microns, and on green barley leaves 
83.14dzO.29 microns. The difference between the means of the spores 
produced on the agar and on the leaves is 37 times as great as the 
probable error of the difference. 

The temperature relations of the fungus were studied and it was 
found that the mycelium will grow at from i° C. to 37 C, the optimum 
lying near 28 . The spores germinated in redistilled water about equally 
well at temperatures ranging from 6° to 39 , but the length of the germ 
tubes indicated that the optimum temperature is between 22 and 32 . 
Germ tubes penetrated the tissue of both the coleoptile and the leaf at 
from 12 to 34 , but severe infection occurred through a narrower 
range, from 22 to 30 , the disease developing faster at the higher 
temperatures. Above 30 , however, the development of the lesions 
seemed to be checked, altho they appeared verv soon after inoculation. 
In general, we may say that rather high temperatures are most favor- 
able to the growth of the fungus, to spore germination, to infection, and 
to the development of the disease. 



46 TECHNICAL BULLETIN 17 

In phosphoric acid-potassium hydroxide solutions, the spores 
germinated through a wide range of hydrogen-ion concentrations. A 
double optimum occurred, both maxima falling on the alkaline side of 
neutrality at pH 8.2 and pH 9.2. In Czapek's solution minus the sugar, 
the maximum germination occurred at pH 6 and pH 8. In general, 
the spores germinate better in alkaline .solutions than in acid solutions. 
The spores will tolerate high degrees of alkalinity. 

Leaf infection increases directly with the amount of moisture 
present. Greenhouse experiments indicate that the effects of root and 
foot infections are more severe in extremely dry and extremely wet 
soils than in soils containing an optimum amount of moisture for the 
growth of the host plant. 

During one year's held experimentation, no correlation was found 
between the fertility of the soil and the development of foot- and 
root-rot. 

The pathogenic effect of 77. sativum isolated from barley plants in 
Minnesota was compared with that of a Helminthosporium isolated 
from stunted wheat in Illinois and with Fusarium culmorum isolated 
from scabby wheat. Experiments were made to determine the ability 
of these organisms to cause root- and foot-rot of Marquis wheat and 
Lion barley. Under the conditions of the experiment, the Helmin- 
thosporiums caused more injury than the Fusarium. The Minnesota 
strain of Helminthosporium caused the greater amount of seedling 
injury on the Lion barley, while the Illinois strain caused the greater 
dwarfing of the mature plants on both wheat and barley. 

As a result of these studies, the wide-spread occurrence of H. 
sativum may be explained by the fact that the fungus responds sapro- 
phvticallv to such a wide range of environmental conditions. Neither 
the effect of temperature nor acidity seems to be a limiting factor in the 
development of the disease so far as spore germination is concerned. 
As a parasite, the fungus causes rather limited local infections. The 
amount of injury is determined largely by the number and size of the 
lesions. A direct correlation exists between the amount of moisture 
present and the number of lesions. The severity of the infection is 
greater at rather high temperatures than at low temperatures. The 
disease may be expected to develop most severely, therefore, at high 
temperatures in the presence of sufficient moisture. 

Root and foot infections are more severe in certain soils than in 
others. This is probably largely due to differences in soil moisture 
and temperature. In general, the disease causes the greatest injury 
under conditions unfavorable to the growth of the host. Factors, such 
as soil fertilitv, which might then be expected to influence the disease, 
apparently have little effect. 



PATHOGENICITY OF H. SATIVUM 47 

LITERATURE CITED 

1. Alway, F. J. Agricultural value and reclamation of Minnesota peat soils. 
Minn. Agr. Exp. Sta. Bui. 188. 1920. (Out of print.) 

2. Babcock, E. B. and Clausen, R. E. Genetics in relation to agriculture. 
New York, 1918. 

3. Christensen, J. J. Studies on the parasitism of Helminthosporium sativum 
P.K.B. Master's Thesis. Minn. Agr. Exp. Sta. Tech. Bui. 11. 1923. 

4. Clark, W. M. The determination of hydrogen-ions. Baltimore, 1920. 

5. Hamblin, C. 0. "Foot-rot" of wheat caused by the fungus Helminthosporium. 
Agr. Gez. N. S. W. 33:13-19. 1922. 

6. Hayes, H. K. and Stakman, E. C. Resistance of barley to Helminthosporium 
sativum P.K.B. Phytopathology 11:405-411. 1921. 

7. Johnson, A. (i. Helminthosporium diseases of barley in Wisconsin. Phytopath. 
3:75- I9I3- 

8. McKinney, H. H. The Helminthosporium disease of wheat and the influence 
of soil temperature on seedling infection. Phytopath, 12:28. 1922. 

9. Pammel, L. H., King, Charlotte M., and Bakke, A. L. Two barley blights, 
with comparison of species of Helminthosporium on cereals. Iowa State 
College of Agr. and Mech. Arts. Bui. 116, June, 1910. 

10. Schroeder, H. Die Widerstandsfalhigkeit des Weizen-und Gerstenkorns 
gegen Figte und ihre Bedeutung fuer die Sterilisation. Centralblrtt fur Bakt. 
P'arasit. und Infekt. 28:492-505. 1910. 

11. Stakman, Louise J. A Helminthosporium disease of wheat and rye. Minne- 
sota Agr. Exp. Sta. Bui. 191. July, 1920. 

12. Stevens, F. L. Foot-rot of wheat. Science N. S. 51:517-518. 1920. 

13. Helminthosporium and wheat foot-rot. Phytopathology 11:37. 1921. 

14. Webb, Robert W. Studies in the physiology of the fungi. X. Germination of 

the spores of certain fungi in relation to hydrogen-ion concentration. Annals 

Missouri Botanical Garden 6:201-222. 1919. 



8°C, 


(3) 13° ■ 


-i5°C 


2 4 °C., 


(6) 30° 


— 32° C, 


A 2°C. 







PLATE I 

Helminthosporium sativum P. K. B. grown on potato dextrose agar at 
the following temperatures : 
(i) o°— 2°C, (2) 6° 
(4) 17° — 22°C, (5) 21° 
(7) 34° — 35°C, (8) 4 o° 
Incubated g drys. 

PLATE II 

Helminthosporium sativum F. K. B. grown on potato dextrose agar 
at the following temperatures : 
(i) 3 °- 6°C, (2) i2 c 
(4) 20° — 23°C, (5) 27 c 

Incubated 7 days. 



I3°C, (3) 15° 
28° C, (6) 3i° ■ 


— i8°C, 

— 33°C. 


PLATE III 





Marquis wheat showing the effect of Helminthosporium root-rot in 
the following types of soil : 

A I. Heavy loam, soil inoculated with //. sativum P.K.B. 

A iC. Heavy loam, uninoculated, sterilized soil 

A 2. Sandy loam, soil inoculated with II. sativum P.K.B. 

A 2C. Sandy loam, uninoculated, sterilized soil 

A3. Sand, soil inoculated with II. sativum P.K.B. 

A 3C. Sand, uninoculated, sterilized soil. 

A 4. Peat, soil inoculated with II . sativum P.K.B. 

A 4C. Peat, uninoculated, sterilized soil. 

PLATE IV 

Lion barley plants, 3 weeks old, growing in soil inoculated with the 
following organisms : 

1. Helminthosporium sativum P.K.B. Culture 82a, isolated from stunted 

barley plants in Minnesota 

2. Helminthosporium isolated from stunted wheat plants in Illinois 

3. Fusarium culmorum ( W. Sm.) Saccardo 

4. Uninoculated, sterilized soil 

PLATE V 

Lion barley plants, 3 weeks old, showing effect of the following soil 
organisms on development of root systems 

1. Helminthosporium sativum P.K.B. Culture 82a 

2. Helminthosporium from stunted wheat plants in Illinois 

3. Fusarium culmorum (W. Sm.) Saccardo 

4. Normal roots grown in uninoculated, sterilized soil 

PLATE VI 

Lion barley plants, 3^ weeks old, showing effect of root infection by 
Helminthosporium sativum P.K.B. The eight seedlings on the left were 
grown in inoculated soil, the one on the right in uninoculated, sterilized soil. 




PLATE I 




PLATE II 



Louise Dosdall was born in Waco, Texas, December II, 
1893. In 1901 her parents moved to LeSueur, Minnesota. 
She attended the public schools there, and then finished the 
grammar schools at St. Paul, Minn. From 1908 to 1912 she 
attended Humboldt High School, in St. Paul. 

In 1912 she entered the College of Science, Literature, and 
the Arts of the University of Minnesota. As an undergraduate, 
she majored in botany, specializing particularly in plant ecology. 
Her minor work was in chemistry and education. In 1916 she 
received the B.A. degree. During the following year she acted 
as teaching fellow in botany at Macalester College, St. Paul, 
and pursued gradurte work in plant ecology and plant pathology 
at the University of Minnesota, receiving the M.A. degree in 
1917. 

From June, 1917, to July, 1920, she was a half-time assistant 
in plant pathology in the College of Agriculture, University of 
Minnesota, devoting the remainder of her time to graduate 
work in plant pathology and mycology. 

Since July 1920 she has been an instructor in the College 
of Agriculture and Mycologist in the Agricultural Experiment 
Station of the University of Minnesota. 



LIBRQRY OF CONGRESS 



002 812 252 4 



